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J. Bacteriol., 08 1995, 4327-4332, Vol 177, No. 15
Copyright © 1995, American Society for Microbiology

Substrate requirements for ErmC' methyltransferase activity

P Zhong, SD Pratt, RP Edalji, KA Walter, TF Holzman, AG Shivakumar and L Katz
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA.

ErmC' is a methyltransferase that confers resistance to the macrolide- lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.

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